A survey of gastrointestinal helminth infestation in smallholder backyard pigs and the first molecular identification of the two zoonotic helminths Ascaris suum and Trichuris suis in Myanmar

Background Parasitic infestations have a substantial economic impact on pig production. This study aimed to investigate the gastrointestinal (GI) helminths in pigs and to molecularly characterise two important nematodes, Ascaris and Trichuris species. Materials and methods A total of 500 pig faecal samples were collected from small holder backyard pig farms in five townships within Nay Pyi Taw, Myanmar. Microscopic examination was conducted to estimate the prevalence of GI helminth infestation in the pigs. DNA extraction and PCR were performed on faecal samples that were morphologically positive for Ascaris and Trichuris eggs. Molecular analysis was then conducted to characterise A. suum and T. suis, the most common and zoonotic helminths. Results According to microscopic examination, 69.2% (346/500) were positive for GI helminth eggs. The GI helminth species observed were A. suum, Strongyle, Strongyloides spp., T. suis, Metastrongylus spp., Hyostrongylus spp., Fasciolopsis spp., Paragonimus spp., and Schistosoma spp., with occurrences of 34.8%, 29.6%, 21.4%, 20.0%, 4.0%, 1.6%, 1.0%, 1.0%, and 0.4%, respectively. Mixed infections of GI helminths were noted in 31.0% of the samples. Overall, sampled pigs excreted mostly low levels (< 100 EPG) or moderate levels (> 100–500 EPG) of GI helminth eggs. The highest mean EPG for each parasite species was noted in A. suum. The presence of A. suum and T. suis was confirmed molecularly. The sequences of the internal transcribed spacer 1 (ITS1) region of A. suum showed high similarity with previously reported sequences. Likewise, the sequences of T. suis exhibited high similarity with the sequences reported from humans and pigs. Age was noted as an associated factor (P < 0.05) for GI helminth infection status. Conclusions In this report, A. suum and T. suis were molecularly identified for the first time in Myanmar. It is important to extend the information among the farmers to be aware of the necessity of preventing zoonotic parasites by practicing regular deworming, proper use of anthelmintics and maintaining hygienic conditions in their pig farms. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-024-03998-w.


Background
Pig productivity is under threat due to a wide range of pig diseases.Infectious diseases have a substantial economic impact on pig production because they can reduce productivity and reproduction, as well as increase morbidity and mortality [1].Parasites are frequently reported as the cause of diseases in pigs, particularly in tropical regions.Among parasitic diseases, gastrointestinal (GI) parasites are responsible for substantial loss of productivity in pigs in terms of inefficient feed conversion, poor growth rate, intestinal malabsorption, reduced weight gain, decreased litter size delayed or incomplete immunity subsequent to vaccinations, negative effects on meat quality and the condemnation of affected organs after slaughter [2].Additionally, several swine parasites can be transmitted from pigs to humans, posing a significant hazard for the producer [3].
The most common helminth infestations in humans and pigs around the world are caused by nematode worms of the genera Ascaris and Trichuris [4].Some parasites, such as Ascaris suum and Trichuris suis, are widespread in pigs and can infect both humans and pigs [4,5].The worms that infect pigs and humans are morphologically similar and difficult to distinguish due to a lack of distinct characteristics [5].Infection with A. suum may result in production losses due to altered carcass composition, lower weight gain, and liver condemnation [6].Furthermore, the level of natural worm crosstransmission between pig and human hosts is unknown; nevertheless, experimental cross-infections have proven that A. suum can infect humans, and human zoonotic cases have been reported [5,7,8].Infection with T. suis (whipworm) in pigs can result in anorexia and bloody diarrhoea in growing pigs, which can lead to economic losses [9].Because of the similarities among Trichuris species, morphological differentiation is very difficult.Moreover, the degree of natural Trichuris cross-transmission between people and pigs is unknown [5].Although adult worms rarely persist, investigations on experimental infection have shown that human whipworm T. trichiura can establish in pigs, whereas patent T. suis infection has been noted in humans [5].Because species discrimination by egg morphology is challenging, the development of molecular approaches for species identification and diversity evaluation is extremely beneficial [10].Several molecular markers have been used for the identification of Ascaris and Trichuris spp., such as nucleotide sequences of internal transcribed spacers 1 and 2 (ITS1 and ITS2) [11,12].
The pig population in Myanmar in 2021 was estimated to be approximately 6.8 million [13].Approximately 86% of the Myanmar population resides in rural areas and is involved in agro-livestock production and agro-industrial work.Most pig farmers in Myanmar prefer backyard farming due to its simplicity and low cost, generally with intensive or semi-intense methods [14].Even though pig production in Myanmar contributes to farmers maintaining a sustainable livelihood, there are many challenges to maintaining productivity, profitability and sustainability.One of these challenges is parasitic infestation.Although our recent study demonstrated the occurrence of Cystoisospora infection in pigs in Myanmar [15], data on helminth infestation are very limited.Therefore, this study aimed to investigate the GI helminths in pigs and to molecularly characterize two important nematodes, Ascaris and Trichuris species, in the Nay Pyi Taw area, Myanmar.

Distribution of GI helminth infestation among age groups, feeding systems, animal housing floors, hygiene conditions and associated factors
The mean EPG was 261.4 (ranging from 16.7 to 13,800) for overall helminth infestation in this study.The mean Conclusions In this report, A. suum and T. suis were molecularly identified for the first time in Myanmar.It is important to extend the information among the farmers to be aware of the necessity of preventing zoonotic parasites by practicing regular deworming, proper use of anthelmintics and maintaining hygienic conditions in their pig farms.Keywords Ascaris suum, Trichuris suis, Backyard pigs, Helminth parasites, Myanmar EPG (Mean ± SD) values in age groups were 1031 ± 2,023 (Median = 225) in weaners, 429 ± 1177 (Median = 90) in growers and 646 ± 1862 (Median = 143) in adults.Among the age groups, the weaner group showed the highest EPG when compared to the other age groups (P < 0.05) (Fig. 2).Although not significantly different, the highest intensity of helminth infection was found in pigs fed local feed (827 ± 1,946 EPG and median = 150), followed by pigs fed mixed feed (573 ± 1640 EPG and median = 1,640) and pigs fed commercial feed (306 ± 264 EPG and median = 330).In this study, a higher intensity of helminth infestation was found in farms with ground floors (799 ± 1,525 EPG and median = 150), and a lower intensity was found in farms with concrete floors (643 ± 1,836 EPG and median = 120).Furthermore, a higher intensity of helminth infestation was found in farms with no hygiene practices (799 ± 1,525 EPG and median = 150) than in farms with hygiene practices (643 ± 1,836 EPG and median = 120) (Fig. S2).According to the chi-square test, age was identified as the only factor associated (P < 0.05) with the occurrence of GI helminth infestations in pigs in this study (P = 0.026, χ 2 = 7.306).Breed, anthelmintic treatment, floor type, feed type and hygienic condition of the farm were not associated with the occurrence of GI helminth infestations.

Molecular identification of Ascaris suum and Trichuris suis
PCR was performed on 15 samples from each of the A. suum-and T. suis-positive samples by microscopic examination.Sequencing was performed on three samples with a targeted size of ~ 515 bp for the A suum-specific ITS1 PCR products.The obtained nucleotide sequences were all clustered together with A. suum sequences available in the database (Fig. 3), with being 99.6 to 100% identical to A. suum from pigs in Japan (AB576592, AB571302 and AB110022), China (HQ721825) and Thailand (MF358944), and one from humans in Lao PDR (MF358943).Three samples were used for sequencing of the T. suis-specific ITS2 PCR products with a targeted size of ~ 635 bp.The obtained nucleotide sequences were all clustered together with T. suis sequences available in the database (Fig. 4), showing 100% identity to the sequences of T. suis from pigs in Egypt (MN967779) and 99.5%, 99.1%, and 98.7% identities to T. suis sequences Fig. 2 The intensity of helminth infestation (EPG) was higher (P < 0.05) in weaners than in growers and adults.The top and bottom horizontal lines of the boxplots represent the first and third quartiles of the data range, respectively.The medians are shown by middle horizontal lines, and the data range is shown by vertical lines, with outliers plotted as points.The notches of each boxplot are approximate 95% confidence intervals of medians Fig. 3 The phylogenetic relationship of partial ITS1 sequences of Ascaris suum detected in this study and reference sequences.The phylogenetic tree was constructed by the maximum likelihood method based on the Tamura-Nei model.The bold taxa represent the sequences obtained from the current study.The GenBank accession number of each sequence is given.Bootstrap values were computed independently for the purposes of 1000 replicates from pigs in China (MG656441, AM993015 and AM993007), Uganda (JN181800), and Spain (AJ249966), respectively (Table 2).

Discussion
Pigs are frequently affected by helminth parasites worldwide in all types of production systems.The findings of this study were the first to reveal the occurrence of GI helminths in pigs in Myanmar as well as the molecular identification of pig helminths.This study found a higher occurrence of 62.9% compared to 56.95% in Rajasthan district, India [16], and 37.5% in Aizawl district of Mizoram, India [17].The differences in the infection rates were attributed to factors such as breed, geographical condition, climate, hygiene and faecal examination techniques [18].The higher occurrence could be attributed to the fact that all of the farms enrolled in this study were smallholder farms with poor hygienic conditions.Many investigations stated that the spread of intestinal helminths in pigs raised using traditional systems was due to poor hygiene, poor nutrition and inadequate anthelmintic interventions [19,20].Among the helminths observed in this study, A. suum was the dominant species, with an occurrence of 34.8% and the highest mean EPG of 173.Although the prevalence varies greatly with the environment and production system, A. suum is the most or second most common intestinal species in farmed pigs globally [6).The higher prevalence of Ascaris sp. was also investigated in Denmark (88%) [21], the Netherlands (72.7%) [22], Nagaland, India (65.5%) [23], Vietnam (51%) [24], Nepal (45.0%) [25], South Africa (44.5%) [26], Burkina Faso (40%) [18], and China (36.7%) [27].A lower prevalence was reported in India (11.1-33.3%)[28,29], Kenya (28.7%) [19], and Rwanda (10.6%) [30].
According to Băieş et al. [31], A. suum is the most common endoparasite of swine in most countries and is one of the most economically important parasites.Thus, the highest occurrence of Ascaris observed in this study should be considered for worm control, although the sampled pigs were apparently healthy.Moreover, the zoonotic potential of Ascaris should also be considered because its occurrence (34.8%) in the present study was high.Among the ten helminth species observed, Strongyloides spp., Trichuris sp., Fasciolopsis spp., and Schistosoma spp.are zoonotic helminths with occurrences of 21.4%, 20%, 1%, and 1%, respectively.Therefore, it is crucial to manage the smallholder pig farms in the Nay Pyi Taw area to lessen the impact of helminth infestations by practicing proper deworming strategies.
According to the statistical analysis, the occurrence of GI helminth infestation was found to be associated with swine age in this study.Pigs < 3 months of age had an increased risk of infestation when compared to older pigs.The other hypothesized factors, breed, anthelmintic treatment, floor type, feed type and hygienic condition of pig farm, appeared to have no association with GI helminth infestation in this study.For the age factor in this study, a higher infestation rate was noted in younger pigs.Immunity to GI helminths might develop in older pigs and thus could be a reason for the higher occurrence in younger pigs.Foster and Elsheikha [41] also pointed out that the lower prevalence of GI parasites in adult pigs could be due to the enhanced resistance and susceptibility to reinfection governed by increased immunological memory.
In the studied townships, the GI helminths' occurrences were different with the minimum (52.0%) in Lewe Township and maximum (85.0%) in Pyinmana Township.However, farm management practices, such as deworming, hygienic conditions, feed type and floor type, were not much different among the townships.The significant association (P < 0.05) could be explained by the fact that sampling in Pyinmana Township was conducted at the end of May when the rainy season starts in Myanmar.In the remaining four townships, sampling was performed in the months of December and January.Parasitic worm eggs and larval development prefer a moist environment, and thus, the parasitic contamination rate increased in pigs and the environment.In accordance with this fact, it could be considered that the highest occurrence was noted in Pyinmana Township.
Overall, sampled pigs excreted mostly low levels (< 100 EPG) and moderate levels (> 100-500 EPG) of GI helminths.Thus, it is clear that the sampled pigs seemed to be normal in clinical appearance without showing any abnormalities due to harbouring moderate levels of GI helminth infestations.However, de Araújo et al. [42] suggested that subclinical infections are important and can be frequent, resulting in loss of appetite, low weight gain, and reduced feed conversion in affected animals.
The eggs of most common worms that infect pigs and humans (A.suum and T. suis) are morphologically similar to other species of the same genera and thus the ITS1 and ITS2 sequences have frequently been used for distinguishing closely related species [43].In this investigation, we identified and examined the sequences of the ITS1 region of A. suum and the ITS2 region of T. suis and compared them to previously published sequences of those infecting humans and pigs from various regions.Sequence analysis of A. suum identified from humans and pigs revealed that the isolates belong to the same clade as the sequences documented in Asian countries and Brazil.Additionally, sequences of T. suis identified from pigs and T. trichiura from humans also belong to the same clade.These findings suggest that humans and pig-derived A. suum exhibit genetic similarities with sequences reported from Asian countries and that T. suis likewise shares genetic similarities between humans and pigs.Therefore, this finding supported the findings of Sadaow et al. [44], who assumed that zoonotic cross-transmission of Ascaris roundworm between pigs and humans might exist in Thailand, Lao PDR, and Myanmar.Although the utilization of ITS1 and ITS2 sequences is beneficial in distinguishing between parasite species [11], their resolution power is comparatively lower than that of other genomewide identification techniques.

Conclusion
The overall GI parasite infestation rate in pigs was 69.2%, with 31.0%mixed infections, and five species of zoonotic helminths were found in the present study.Furthermore, age was a major factor related to GI helminth infestation in smallholder backyard pigs in the Nay Pyi Taw area.The two zoonotic helminths, A. suum and T. suis, were molecularly identified for the first time.It is important to extend the information among the farmers to be aware of the importance of prevention of zoonotic parasites for public health by practicing regular deworming, proper use of anthelmintics and keeping hygienic conditions in their pig farms.

Sample size and sample collection
The study area, sample size and sampling period were reported in our previous study [15].In brief, a crosssectional study was conducted in five townships within Nay Pyi Taw, located between latitude 19° 45′ N and longitude 96° 06′ E, between December 2020 and May 2021.Samples were collected mostly in suburban and rural areas within townships, which were home to a large number of pig farms.A total of 500 fresh faecal samples were obtained, with 100 samples from each township.Upper parts of freshly dropped faeces on the ground of individual pigs were collected, placed into individual zip lock bags, labelled, put in an ice box, and brought to the Laboratory of Department of Pharmacology and Parasitology, University of Veterinary Science.During sampling, information regarding the age, sex, breed, type of feed, floor type and hygienic condition of the pig farm, as well as anthelmintic usage, were all recorded.The pig breeds included local and DYL (a cross breed of Duroc, Yorkshire, and Landrace).According to Esrony et al. [45], animals were classified as weaners (5-12 weeks), growers (> 12 weeks to 24 weeks), and adults (> 24 weeks).

Laboratory analysis for parasite eggs
The samples were examined for helminth parasites in the laboratory using the faecal flotation and sedimentation methods as described by Zajac and Conboy [46].On the basis of morphological characteristics described by Taylor et al. [47], parasite species were identified.The McMaster egg counting method was used to calculate the number of eggs per gram (EPG) of faeces [46].

DNA extraction and PCR
DNA extraction was performed on faecal samples that were positive for A. suum or T. suis eggs by fecal examination.According to the manufacturer's instructions, DNA was extracted using the Power Fecal DNA Isolation kit (MO BIO Laboratories, USA).The extracted DNA samples were eluted in 200 μl elution buffer and kept at -80 °C.A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA) was used to determine the DNA concentration.To amplify the ITS1 region of A. suum, a primer set consisting of the forward primer F2662 (5'-GCA AAA GTC GTA ACA AGG T-3') and the reverse primer R3214 (5'-CTG CAA TTC GCA CTA TTT ATCG-3') was employed [11].For the amplification of the ITS2 region of T. suis, a primer set of the forward primer ITS2_tt_F2 (5'-GCT CGT AGG TCG TTG AAG -3') and the reverse primer ITS2_tt_R2_new1 (5'-GGG CAG CTT CCG TACT-3') was used [12].Thermal cycling began with denaturation at 94 °C for 1 min, then 40 cycles at 98 °C for 10 s, 52 °C (for A. suum) and 54 °C (for T. suis) for 15 s, 68 °C for 1 min, and a final extension at 68 °C for 5 min.DNA samples from previously collected A. suum and T. suis specimens were used as positive controls, with molecular grade deionised water serving as negative controls.PCR products were examined using 2% Tris-acetate-EDTA (TAE) agarose gel electrophoresis after staining with RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology Inc., Seongnam, Korea).

Sequencing and phylogenetic analysis
Following the manufacturer's instructions, DNA fragments obtained from the PCR were excised from the gel and purified using a NucleoSpin ® Gel and PCR Clean-up Kit (MACHEREY-NAGEL, Düren, Germany) and submitted for direct sequencing on an Applied Biosystems 3130 Genetic Analyzer with a BigDye v3.1 Terminator cycle sequencing kit (Applied Biosystems, Inc., Carlsbad, CA, USA).ATGC version 7 (GENETYX Corporation, Tokyo, Japan) was used for multiple sequence alignment.The phylogenetic analysis was conducted using the maximum likelihood (ML) method in MEGA X with Tamura-Nei model [48].The bootstrap analysis was performed with 1000 replicates per tree.The obtained sequences (OQ825946-OQ825951) were compared to those in the NCBI nucleotide database (http:// www.ncbi.nlm.nih.gov/ nucco re/).

Statistical analysis
All data were entered into Microsoft Excel 2013 and analysed using the Statistical Package for Social Science (SPSS) version 20.0.The association between the occurrence of GI helminths and the hypothesized factors of age, sex, breed of pigs, anthelmintic treatment, floor type, feed type, and hygienic condition of the pig farm was analysed using a Pearson's Chi-square test at the P < 0.05 level of significance.The boxplots were explored by the ggplot package using the R language platform [49,50].

Table 1
Occurrence of GI helminth infestation in pigs in Nay PyiTaw area (n = 500) Fig. 1 EPG of the most common GI helminth species (Ascaris sp., Strongyle, Trichuris sp. and Strongyloides spp.) observed in five townships

Table 2
Identification of sequences of Ascaris suum or ITS2 sequences of Trichuris suis isolates from pigs in Myanmar using the NCBI BLAST search